The myosin family of molecular motors plays crucial roles in contraction, diverse forms of cell movement, and changes in cell shape. Myosin II is a non-processive motor that drives cytokinesis and muscle contraction, and myosin V and VI are processive motors that drive vesicular movements in neurons and other cells. The molecular basis of myosin function has reached a detailed level of analysis, due to the advent of in vitro motility assays, total internal reflection fluorescence microscopy for measuring movement of single molecules, and the establishment of laser trap assays that allow the direct measurement of force and displacement produced by a single myosin molecule pulling on a single actin filament. The specific aims of this proposal are to continue to develop these assays, and to use them and other approaches to examine the mechanical and biochemical parameters of native and mutated forms of both myosin V and myosin VI. We will also initiate studies on other members of the myosin family. Specific goals involving myosin V include preparation and characterization of a two-headed construct of myosin V joined by a totally flexible hinge, to test the role of the proximal "hip" region in processive movement. We will visualize individual fluorescent nucleotides as they come on and off myosin V during the stepping process to test models of chemomechanical coupling. These studies will be complemented with determination of the effects of both backward and forward external loads on the behavior of myosin V stepping. Similar studies will be carried out with myosin VI and other myosin family members, including preparation of altered forms of the motors to examine the roles of various domains. A primary goal for this grant period is to determine the molecular basis of movement of myosin VI, which is a non-classical myosin that moves by an unknown mechanism, but may involve the melting out of some part of the protein in a nucleotide dependent manner. We will use multiple approaches to reveal regions that become exposed during its chemomechanical cycle.